Wolfson Centre for Age-Related Diseases, Institute of Psychology, Psychiatry & Neuroscience, King's College London, London, UK.
Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
Mol Pain. 2024 Jan-Dec;20:17448069241293286. doi: 10.1177/17448069241293286.
The synaptic connections between dorsal root ganglia (DRG) and dorsal horn (DH) neurons are a crucial relay point for the transmission of painful stimuli. To delineate how synaptic plasticity may modulate the excitability of DH neurons, we have devised a microfluidic co-culture model that recapitulates the first sensory synapse using postnatal mouse sensory neurons. We show that DRG-DH co-cultures characterize salient features of the in vivo physiology of sensory neurons. Immunocytcochemical experiments of the cultured DH neurons show a co-localization of Map2 with VGlut2 and of Map2 with Synapsin 1, corroborating the glutamatergic identity of the DH neurons and further suggesting the potential formation of active synapses in this neuronal set. Fluorometric imaging experiments demonstrate the elicitation of calcium responses in DH neurons following the stimulation of DRG cell bodies or axons. Selective NMDA and AMPA receptor blockade appreciably silences DH neuron responses, suggesting that glutamatergic signaling is maintained in vitro. Last, a surrogate model of peripheral nerve injury is introduced in the form of an axotomy, which results in elevated and prolonged calcium responses of DH neurons. Overall, the microfluidic mouse co-cultures provide a method advancement in the study of periphery-to-center pain signaling, where the potential of utilizing the platform for drug target identification is underscored.
背根神经节 (DRG) 和背角 (DH) 神经元之间的突触连接是疼痛刺激传递的关键中继点。为了阐明突触可塑性如何调节 DH 神经元的兴奋性，我们设计了一种使用产后小鼠感觉神经元的微流控共培养模型来再现第一个感觉突触。我们表明，DRG-DH 共培养物具有感觉神经元体内生理学的显著特征。培养的 DH 神经元的免疫细胞化学实验显示 Map2 与 VGlut2 和 Map2 与 Synapsin 1 的共定位，证实了 DH 神经元的谷氨酸能特性，并进一步表明在该神经元集中可能形成活性突触。荧光成像实验表明，刺激背根神经节细胞体或轴突后，DH 神经元会引发钙反应。选择性 NMDA 和 AMPA 受体阻断可显著沉默 DH 神经元反应，表明谷氨酸能信号在体外得到维持。最后，以轴突切断的形式引入了外周神经损伤的替代模型，这导致 DH 神经元的钙反应升高和延长。总体而言，微流控小鼠共培养物为研究外周到中枢疼痛信号提供了一种方法上的进步，强调了该平台在药物靶点识别中的应用潜力。
